Structural, mechanistic and functional characterization of glycoside hydrolases of family GH99

Glycosylation is a very common post-translational modification and the glycans can be attached to oxygen (O-linked), nitrogen (N-linked) or carbon (C-linked). N-linked glycosylation has implications for protein folding and is also essential in viral infectivity and cell-cell signalling. Endo-α-1,2-mannosidase from family GH99 is a unique enzyme within the N-glycosylation pathway as it is the only one which does not cleave the terminal sugar from the reducing end of the glycan, but instead releases an α-Glc-1,3-Man disaccharide, with overall retention of stereochemistry at the anomeric carbon. Previously it was proposed that GH99 endo-acting mannosidases and mannanases proceed through a neighbouring group participation mechanism with a 1,2-anhydrosugar as a reaction intermediate. This Thesis contains evidence supporting this hypothesis. Chapter 2 presents structures of the bacterial GH99 with its substrate, with mimics of the reaction intermediate and with the products of the reaction. Kinetic and structural data on various intermediate mimics show that the compound whose structure is the closest to the intermediate is turned over by the enzyme. In Chapter 3, analysis of different designs of GH99 inhibitors and their conformation on-enzyme is presented. Chapter 4 presents purification and solution of the crystal structure of the catalytic domain of the human endomannosidase (MANEA). Multiple crystal forms were obtained, which made it possible to look at the conformation of a feature present in the eukaryotic but not bacterial GH99: a loop spanning residues 191–201. This loop was disordered when no ligand was present in the –2/–1 sites, and ordered when these sites were occupied. Chapter 5 explores attempts at producing MANEAL, a paralog of MANEA which is found in bony vertebrates. The Thesis concludes with an analysis of the phylogeny of endomannosidase genes and perspectives for future research: studies of endomannosidase in mammalian model organisms are needed to understand its significance.